Epidermal Growth Factor Receptor (EGFR) is a member of the type 1 tyrosine kinase family of growth factor receptors, which play critical roles in cellular growth, differentiation, and survival. Activation of these receptors typically occurs via specific ligand binding, resulting in hetero- or homodimerization between receptor family members, with subsequent autophosphorylation of the tyrosine kinase domain. This activation triggers a cascade of intracellular signaling pathways involved in both cellular proliferation (the ras/raf/MAP kinase pathway) and survival (the PI3 kinase/Akt pathway). Members of this family, including EGFR and HER2, have been directly implicated in cellular transformation.
The EGFR signaling pathway is activated in several cancers, such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC), head and neck cancer and gliomas. Activation may occur by multiple mechanisms, including activating mutations in the EGFR protein, or EGFR overexpression, typically due to increased EGFR gene copy number. The EGFR protein expression can be assessed semi quantitatively by immunohistochemistry. Gene copy number can be evaluated by several methods, including in-situ hybridization and gene mutations can be detected by several methods including direct sequencing.
EGFR antibodies in clinical use (e.g., cetuximab (ERBITUX™) and panitumumab (VECTIBIX™)) bind to the extracellular domain of the EGFR. This receptor domain includes the ligand binding site and these antibodies are believed to block ligand binding; thereby, disrupting EGFR signaling. As a result of the therapeutic utility of such EGFR antibodies, many subsequent studies have focused on the production of antibodies (or other binding molecules) specific for the EGFR extracellular domain (see, e.g., U.S. Pat. Nos. 5,459,061, 5,558,864, 5,891,996, 6,217,866, 6,235,883, 6,699,473, and 7,060,808; European Pat. Nos. EP0359282 and EP0667165).
Approximately 10-20% of unselected CRC patients respond to anti-EGFR antibody therapy. In CRC, as in many other cancers, neither the diagnostic characteristics of the tumor nor the degree of EGFR expression evaluated by immunohistochemistry, are thought to correlate with clinical response to anti-EGFR antibodies, such as cetuximab, matuzumab (hMab 425) or panitumumab. Currently, therefore, most treated patients are exposed to the risk of ineffective therapy with undesired side effects.
KRAS gene mutational status can predict the response to the anti-EGFR monoclonal antibodies cetuximab and panitumumab (Allegra, 2009). Tumors harbouring activating mutations of KRAS, a signaling molecule downstream of EGFR, do no benefit from anti-EGFR therapy (Linardou, 2008). In KRAS wild type (WT) patients, on the other hand, the addition of cetuximab to cytotoxic treatment improves the response rates with 16 to 24% compared to cytotoxic therapy alone. About 40% of the KRAS WT patients are non-responders to combination therapy (Bokemeyer, 2009; Van Cutsem, 2009) and a significantly larger fraction of patients are non-responders to EGFR antibody monotherapy (Amado, 2008).
In addition to KRAS mutations, changes in other molecules downstream of EGFR, in particular BRAF gene mutations, PIK3CA mutations and loss of expression of the PTEN tumor suppressor protein appear to associate with resistance to anti-EGFR treatment (Laurent-Puig, 2009; Siena, 2009). Accordingly, BRAF testing was recently included in the NCCN Clinical Guidelines in Oncology for Colon Cancer and Rectal Cancer (Engstrom, 2009). However, even the combination of these tests is likely to identify only a minority of non-responsive KRAS WT patients (Laurent-Puig, 2009).
In previous studies the EGFR protein expression level assessed by immunohistochemistry (IHC) has not correlated with response to anti-EGFR antibody treatment (Cunningham, 2004; Saltz, 2004; Chung, 2005). Instead, an increased EGFR gene copy number (GCN) has in some studies shown an association with a favorable response among KRAS WT patients (Sartore-Bianchi, 2007; Cappuzzo, 2008; Lievre, 2006; Moroni, 2005). Fluorescence in situ hybridization (FISH) technique has been used in most previous studies (Moroni, 2005; Cappuzzo, 2008; Personeni, 2008; Scartozzi, 2009; Sartore-Bianchi, 2007). The FISH results are challenging to interpret and the lack of standardization of analytical method and scoring systems may partly explain why the EGFR GCN evaluation has not been incorporated into the clinical practice yet. In fact, the current NCCN colorectal cancel guidelines do not recommend routine EGFR testing, and state that no patient should be either considered or excluded from cetuximab or panitumumab therapy on the basis of EGFR test results (Engstrom, 2009).
In summary, there is a need to explain the differential response in patients to anti-EGFR monoclonal antibodies and to develop a strategy to identify cancer patients such as colorectal cancer patients likely to benefit from or be responsive to anti-EGFR antibody therapy.
US2008/0090233 (Garcia et al.) discloses a method to select a cancer patient who is predicted to benefit or not benefit from therapeutic administration of an EGFR inhibitor. The method is based on the detection of a level of amplification and polysomy of the EGFR gene and the HER2 gene. The assay for detecting gene copy number is based on fluorescence in situ hybridization (FISH).
US2009/0269344 (Siena et al.) discloses an in vitro method for detecting and analyzing whether a patient suffering from a cancer, which overexpresses EGFR, responds positively to the administration of an anti-EGFR antibody. The method comprises the steps of determining the EGFR gene copy number in tumor cells obtained from a patient and selecting said patient for administration with said anti-EGFR antibody, if the tumor cells of the patient display an amplified copy number of the EGFR gene. The assay for detecting gene copy number is based on fluorescence in situ hybridization (FISH).
Hanawa et al. (2006) analyzed EGFR protein expression with IHC and EGFR gene copy number with FISH in cancer samples of esophagus.
Hemmings et al. (2009) analyzed EGFR protein expression using IHC in colorectal cancer samples. They also used CISH to detect gene copy number of EGFR.
Sholl et al. (2009) used IHC to detect EGFR protein in lung adenocarcinoma samples. They also used FISH and CISH to detect EGFR gene copy number and correlated FISH analysis results to those of CISH.
Gaiser et al. (2009) compared the concordance between SISH and FISH methods in glioblastoma patients and used EGFR IHC to detect EGFR protein.
Miyanaga et al. (2008) used IHC to detect EGFR protein expression and CISH method to analyze EGFR gene copy number.
However, none of the above-mentioned prior art documents discloses enzymatic metallography method (e.g. SISH) to detect EGFR GCN and EGFR IHC to select cancer patients for EGFR inhibitor treatment. Further, the cited prior art do not teach that it would be advantageous to determine the area of highest expression of EGFR in a tumor sample by IHC, and then use said area of highest expression in enzymatic metallography to determine gene copy number of EGFR gene or chromosome 7. This approach renders results more reliable and thus EGFR GCN evaluation may become part of clinical practice.